37

Słupskie Prace Biologiczne

Nr 13 ss. 37-54 2016

ISSN 1734-0926 Przyjęto: 7.11.2016

© Instytut Biologii i Ochrony Środowiska Akademii Pomorskiej w Słupsku Zaakceptowano: 16.01.2017

ANTIMICROBIAL SCREENING OF THE VARIOUS

EXTRACTS DERIVED FROM THE LEAVES AND

PSEUDOBULBS OF COELOGYNE SPECIOSA

(BLUME) LINDL. (ORCHIDACEAE)

Lyudmyla Buyun1

Halyna Tkachenko2

Zbigniew Osadowski2

Anna Góralczyk2

Lyudmyla Kovalska1

Oleksandr Gyrenko1

1 M. Gryshko National Botanical Garden

National Academy of Science of Ukraine

Timiryazevska St. 1, Kiev 01014, Ukraine

e-mail: buyun@nbg.kiev.ua 2 Pomeranian University in Słupsk

Institute of Biology and Environmental Protection

Department of Zoology and Animal Physiology

Arciszewski St. 22b, 76-200 Słupsk, Poland

ABSTRACT

The present study was conducted to investigate in vitro antimicrobial activity of

various extracts obtained from vegetative parts of Coelogyne speciosa against Gram-

positive (Staphylococcus aureus ATCC 25923) and Gram-negative bacteria (Esche-

richia coli ATCC 25922). The leaves and pseudobulbs of C. speciosa plants, culti-

vated under glasshouse conditions, were sampled at M.M. Gryshko National Botani-

cal Garden (NBG), National Academy of Science of Ukraine. Freshly collected

leaves and pseudobulbs were washed, weighted, crushed, and homogenized in 96%

ethanol, methanol, ethyl acetate, hexane, and dichloromethane (in proportion 1:19)

at room temperature. Antimicrobial activity was determined using the agar disk dif-

fusion assay (Bauer et al. 1966). The present study has shown that ethanolic extracts

from leaves and pseudobulbs of C. speciosa exhibited strong activity against S. au-

reus (inhibition zone diameter were 21.5 mm and 19 mm, respectively), while meth-

anolic extract from leaves and pseudobulbs revealed mild activity (8.1 and 8 mm).

Moreover, it has been observed that ethyl acetate, hexane and dichloromethane ex-

tracts obtained from leaves and pseudobulbs of C. speciosa revealed no antibacterial

38

activity against S. aureus. Our results also showed that ethanolic extract from leaves of

C. speciosa exhibited strong activity against E. coli (inhibition zone diameter was 21

mm), while other extracts from pseudobulbs revealed minimum activity (inhibition

zone diameter was 12 mm). Those extracts in ethyl acetate, hexane and dichloro-

methane both from the leaves and pseudobulbs revealed no antibacterial activity

against S. aureus. Thus, the research showed that ethanolic extracts of C. speciosa pos-

sess antibacterial potency against S. aureus and E. coli and may be used as natural an-

tiseptics and antimicrobial agents in medicine and veterinary practice. Considering the

medicinal importance of the tested microorganisms, the findings of this research are

considered to be very promising in the perspective of new drug development from

plant sources.

Key words: Coelogyne speciosa Lindl., Staphylococcus aureus, Escherichia coli,

antimicrobial activity, orchids, agar disk diffusion assay

INTRODUCTION

Living plant collections in the botanic gardens are underutilized worldwide re-

sources for plant conservation (Cibrian-Jaramillo et al. 2013). Beside, many groups

of plants are valuable sources of diverse compounds, possessing broad spectrum of

biological activity. In particular, orchids have been used as a source of medicines for

millennia to treat different diseases and ailments including tuberculosis, paralysis,

stomach disorders, chest pain, arthritis, syphilis, jaundice, cholera, acidity, eczema,

tumour, piles, boils, inflammations, menstrual disorder, spermatorrhea, leucoderma,

diarrhoea, muscular pain, blood dysentery, hepatitis, dyspepsia, bone fractures, rheu-

matism, asthma, malaria, earache, sexually transmitted diseases, wounds and sores.

In addition, many orchidaceous preparations are used as emetic, purgative, aphrodis-

iac, vermifuge, bronchodilator, sex stimulator, contraceptive, cooling agent and rem-

edies in scorpion sting and snake bite (Hossain 2011). Pharmacological studies have

revealed antimicrobial, antioxidant, hepatoprotective, anti-inflammatory, anti-arthritic

and wound healing properties of some orchids in pre-clinical studies (Bulpitt 2005,

Panda and Mandal 2013).

Orchids belong to the largest family of angiosperms, with approximately 880

genera and 27,800 species (Givnish et al. 2016). Today, nearly 50 orchid species are

widely used in different systems of medicine (Panda and Mandal 2013). Some of the

species like Vanda tessellata (Roxb.) Hook. ex G. Don, Dactylorhiza incarnata

subsp. incarnata, Dendrobium nobile Lindl. have been already documented for their

proven medicinal values (Kong et al. 2003). Pseudobulbs are most commonly used

to cure ailments, followed by leaves, roots, tubers/rhizomes and flowers. Major local

uses include aphrodisiacs, energizers, and treatments of skin burns, fractured or dis-

located bones (both of humans and cattle), headaches, fever, and wounds. Other uses

include insect repellent, blood purifier, skin fungi, antidote against snake bites and

scorpion stings, inducement of abortions and recovery from child birth. Orchids are

mainly used as paste, powder or juice, solely or mixed with milk, honey or wheat

flour. Orchid extracts are either consumed orally or applied externally. Fresh orchid

39

flowers are used to induce vomiting by exposure to a dominant foul smell. Local

communities in India and Nepal also commonly eat freshly cut species of Coelogyne

in the forest when they feel thirsty (Subedi et al. 2011).

A wide range of chemical compounds has been identified from medicinal orchids

(Subedi et al. 2011). Studies have reported the isolation of huge number of important

phytochemicals from different genera of orchids such as alkaloids, flavonoids, stil-

benoids, anthocyanins, triterpenoids, orchinol, hircinol, cypripedin, bibenzyl deriva-

tives, phenanthrenes, jibantine, nidemin and loroglossin which are present in leaves,

pseudobulb, roots, flowers or in the entire plant (Pant 2013).

Coelogyne speciosa (Blume) Lindl. (Photos 1 and 2) belongs to the section Spe-

ciosae, distributed from mainland Southeast Asia (Thailand), all over Malesia to the

islands in the Pacific Ocean (Gravendeel 2000).

The epithet speciosa (which is Latin for beautiful) refers to the showy flowers.

Pseudobuls are ovoid and clustered, obtusely 4-angled when young. Leaves are one or

two per pseudobulbs. Leaf blade is obovate-lanceolate to linear-lanceolate to lanceo-

late; apex acuminate or cuspidate; main nerves 3-5 (Gravendeel 2000). Synanthous in-

florescences, characteristic feature for C. speciosa, are predominant in the section

Speciosae: the inflorescence-bearing shoot has an immature pseudobulb hidden in the

basal scales and the young leaf or leaves on top of this pseudobulb start swelling and

leaf or leaves fully develop (Gravendeel and de Vogel 1999) (Figs 1 and 2).

The flowers open in succession (starting with the basal one) (www.orchidspecies.

com/coelogynespeciosa.htm).

Artificial pollination treatments, which we carried out under glasshouse condi-

tions, have shown that Coelogyne speciosa is largely self-sterile. Barbara Graven-

deel in Holland has initiated detailed research into Coelogyne speciosa, backed by

extensive DNA analysis (Gravendeel and de Vogel 1999, Gravendeel 2000).

Photo 1. A specimen of Coelogyne speciosa Lindl., cultivated at M.M. Gryshko National Bo-

tanical Garden (Kyiv, Ukraine)

Source: own research

40

Photo 2. The general view of Coelogyne speciosa flower

Source: own research

Moreover, many species of Orchidaceae family also possess antimicrobial activi-

ty (Pérez Gutiérrez 2010).

Fig. 1. The branching pattern of shoot system

of Coelogyne speciosa

Source: own research

Fig. 2. Scheme of the structure of C. speci-

osa elementary shoot: a) leaf; b) pseudo-

bulb; c) buds; d) rhizome; e) node; f) inter-

node

Source: own research

41

Therefore, the aim of this study was to evaluate the antimicrobial activity of the

crude extracts obtained from leaves and pseudobulbs of C. speciosa prepared in dif-

ferent solvent systems against Staphylococcus aureus, a clinically important micro-

organism responsible for many infections, as well as against Escherichia coli to sup-

port the use of these extracts as novel natural products for compounded bacterial-

treatment modalities.

MATERIALS AND METHODS

Collection of Plant Material. The leaves and pseudobulbs of C. speciosa plants,

cultivated under glasshouse conditions, were sampled at M.M. Gryshko National

Botanical Garden (NBG), National Academy of Science of Ukraine. Since 1999, the

whole collection of tropical and subtropical plants (including orchids) has the status

of a National Heritage Collection of Ukraine. Besides that, NBG collection of tropi-

cal orchids was registered at the Administrative Organ of CITES in Ukraine (Minis-

try of Environmental Protection, registration No. 6939/19/1-10 of 23 June 2004).

Preparation of Plant Extracts. Freshly collected leaves and pseudobulbs were

washed, weighted, crushed, and homogenized in 96% ethanol, methanol, ethyl ace-

tate, hexane, and dichloromethane (in proportion 1:19) at room temperature. Conse-

quently five kinds of solvent systems were used to extract the active ingredients

from the leaves and pseudobulbs of Coelogyne speciosa.

Bacterial Growth Inhibition Test of Plant Extracts by the Disk Diffusion Method.

Antimicrobial activity was determined using the agar disk diffusion assay (Bauer et

al. 1966). Cultures of Staphylococcus aureus (ATCC 25923) and Escherichia coli

(ATCC 25922) were suspended in sterile solution of 0.9% normal saline and the tur-

bidity adjusted equivalent to that of a 0.5 McFarland standard. Culture was inoculat-

ed onto Mueller-Hinton (MH) agar plates. Sterile filter paper discs impregnated with

50 μL of extract dilutions were applied over each of the culture plates. Isolates of

bacteria were then incubated at 37oC for 24 h. The plates were then observed for the

zone of inhibition produced by the antifungal activity of C. speciosa. As negative

control, disc impregnated with 50 μl of sterile ethanol, methanol, ethyl acetate, hex-

ane, and dichloromethane was used in each experiment. The antimicrobial activities

of the extracts tested were evaluated at the end of the inoculation period by measur-

ing the inhibition zone diameter around each paper disc in millimeters. The plates

were observed and photographs were taken. For each extract six replicate trials were

conducted against each organism. Zone diameters were determined and averaged.

Statistical analysis. All statistical calculations were performed on separate data

from each bacterial strains (Zar 1999). All statistical calculation was performed on

separate data from each individual with STATISTICA 8.0. The following zone di-

ameter criteria were used to assign susceptibility or resistance of bacteria to the phy-

tochemicals tested: Susceptible (S) ≥ 15 mm, Intermediate (I) = 11-14 mm, and Re-

sistant (R) ≤ 10 mm (Okoth et al. 2013).

42

RESULTS

The present study has shown that ethanolic extracts from the leaves and pseudo-

bulbs of C. speciosa exhibited strong activity against S. aureus (inhibition zone di-

ameter were 21.5 mm and 19 mm, respectively), while methanolic extract from

leaves and pseudobulbs revealed mild activity (8.1 and 8 mm) (Figs 3 and 4). More-

over, it has been observed that ethyl acetate, hexane and dichloromethane extracts

obtained from leaves and pseudobulbs of C. speciosa revealed no antibacterial activ-

ity against S. aureus.

Fig. 3. Antimicrobial activity of various extracts obtained from the leaves of C. speciosa

against Staphylococcus aureus measured as inhibition zone diameter (n = 6)

Source: own research

Fig. 4. Antimicrobial activity of various extracts obtained from pseudobulbs of C. speciosa

against Staphylococcus aureus measured as inhibition zone diameter (n = 6)

Source: own research

43

The results of antimicrobial activity of various extracts obtained from the leaves and pseudobulbs of C. speciosa against E. coli are presented in Figs 5 and 6. Our re-

sults also showed that ethanolic extract from leaves of C. speciosa exhibited strong

activity against E. coli (inhibition zone diameter was 21 mm) (Fig. 5), while others

extracts from pseudobulbs revealed minimum activity (inhibition zone diameter was

12 mm) (Fig. 6). Those extracts in ethyl acetate, hexane and dichloromethane both

from the leaves and pseudobulbs revealed no antibacterial activity against S. aureus

(Figs 5 and 6).

Fig. 5. Antimicrobial activity of various extracts obtained from leaves of C. speciosa against

Escherichia coli measured as inhibition zone diameter (n = 6)

Source: own research

Fig. 6. Antimicrobial activity of various extracts obtained from pseudobulbs of C. speciosa

against Escherichia coli measured as inhibition zone diameter (n = 6)

Source: own research

44

Detailed data regarding the zones of inhibition by the various plant extracts were

recorded and presented in Figs 7 and 8.

A B

Fig. 7. Antibacterial activity of ethanolic extracts obtained from leaves (A) and pseudobulbs

(B) of C. speciosa against S. aureus measured as inhibition zone diameter

Source: own research

A B

Fig. 8. Antibacterial activity of ethanolic extracts obtained from leaves (A) and pseudobulbs

(B) of C. speciosa against E. coli measured as inhibition zone diameter

Source: own research

DISCUSSION

In the present study, the effect of extracts obtained from leaves and pseudobulbs

of C. speciosa prepared in various solvents on the growth of S .aureus and E. coli was investigated in vitro. The results revealed the antimicrobial potential of ethanol-

ic extracts. All the test organisms were susceptible to ethanolic extracts obtained

from leaves and pseudobulbs of C. speciosa with inhibition zone diameter between

12-21.5 mm. E. coli was more resistant to ethanolic extracts obtained from leaves

and pseudobulbs of C. speciosa and the diameter of inhibition zone around the rest

ranged from 11-13.5 mm. The results obtained from the present study are consistent with earlier reports,

which found that other species of Coelogyne have noticeable phytochemical, antimi-

45

crobial, antioxidant and anticancer activity (Sahaya Shibu et al. 2013). According to Sahaya Shibu and co-workers (2013), C. nervosa exhibits potential antimicrobial, antioxidant and anticancer properties. The previous investigations on pharmacologi-cal properties of orchid species, belonging to Coelogyne genus, have found that these plants are sources of phytochemicals which are able to initiate different biolog-ical effects including antimicrobial activity (Pérez Gutiérrez 2010, Sarmad Moin et al. 2012, Sahaya Shibu et al. 2013, Pramanick 2016).

Preliminary phytochemical screening for the presence of alkaloids, carbohy-drates, glycosides, saponins, terpenoids, steroids, flavonoids, phenolic compounds, protein, gum and mucilages, phytosterol, tannins and phlobatannins were carried out. The screening of antimicrobial, antioxidant and anticancer activities was also performed. Preliminary phytochemical analysis revealed that the ethanol and aque-ous extracts shown the maximum phytochemical constituents. This analysis shows the presence of alkaloids, carbohydrates, glycosides, saponins, terpenoids, steroids, flavonoids, phenolic compounds, protein, phytosterol, tannins and phlobatannins in ethanol extract. The result obtained is similar to the results of Sarmad and co-workers (2012) and Mazumder and co-workers (2010) in Coelogyne stricta and Pa-pilionanthe teres. All the extracts of C. nervosa were tested for antimicrobial and an-tioxidant activities. The ethanolic extract of C. nervosa showed the maximum zone of inhibition against the bacteria Pseudomonas aeruginosa (15 mm), Enterococcus faecalis (14.3 mm), Salmonella enterica (12 mm), Bacillus subtilis (11 mm) and Corynebacteria spp. (9 mm). The ethanolic extract showed the maximum antibacte-rial activity against all the microorganisms tested, which shows the minimum inhibi-tory concentration (MIC) value in the range between 400 to 600 (μg/ml). The water extract possessed strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC50 126 μg/ml) and it shows cytotoxic activity towards the MCF-7 cancer cell line (IC50, 292.8 μg/ml). Several compounds like 9,10-Dihydro-5H-phenanthro-(4,5 bcd)-pyrans and pyrones were isolated from a number of species like Coelo-gyne, Pholidota, and Otochilus (Veerraju et al. 1989) whereas, trans-3,4,30,50-tetra-hydroxystilbene is reported to possess a chemopreventative agent with anti-leukemic activity and is being extensively studied for various cancers including colorectal and lung cancer as well as in cardiovascular diseases (Wolter et al. 2002). The anticancer activity results states that the IC50 value of water extract on MCF-7 cell line was 292.8 μg/ml which shows the plant have anticancer activity (Sahaya Shibu et al. 2013).

In a study conducted by Paulomi and co-workers (2013), the bioactivity of few unexplored orchids (Aerides odorata, Acampe papilosa and Acampe ochracea that are native to North-East India) against kanamycin and ampicillin resistant E. coli was found out. All the three plants showed good antibacterial property against the selected strains of E. coli. Among the three strains, the ampicillin resistant variety showed strong inhibition (average zone diameter of 7.0 mm), the kanamycin re-sistant variety showed comparatively less inhibition (average zone diameter of 5.3 mm) and the (MTCC) variety showed the least (average zone diameter of 5.0 mm). The treatment in the antibiotic suspended medium showed less inhibition for acetone extract (average zone diameter of 5.3 mm) against ampicillin resistant variety than water extract (average zone diameter of 6.3 mm) against kanamycin resistant variety (Paulomi et al. 2013).

46

The antimicrobial effect showed by C. speciosa is in agreement with previous an-

timicrobial effects produced by numerous orchids. To screen for bioactive chemicals,

Subedi and co-workers (2011) have tested the extracts of traditionally used plant parts

(either stems or pseudobulbs) of a subset of the orchids used in traditional medicines

or involved in trade for their antibacterial activity against common intestinal and

harmful bacteria (Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus) us-

ing both a disc diffusion method and Minimum Inhibitory Concentration Assay (MIC).

Results of Subedi and co-workers (2011) suggest that traditional uses of wild orchids

against infectious diseases by local communities are based on antibacterial properties

which could be used for the development of alternative drugs. All the extracts showed

activity against one or more test bacteria. Aerides multiflora, Calanthe puberula, Coe-

logyne flaccida, Coelogyne nitida, Coelogyne punctulata, Coelogyne stricta, Cymbidi-

um iridioides, Dendrobium eriiflorum, Dendrobium fugax (syn. Flickingeria fugax), Luisia trichorrhiza and Pholidota imbricata showed a broad spectrum antibacterial ac-

tivity. In contrast, Bulbophyllum umbellatum, Coelogyne flaccida and Malaxis acu-

minata reduced growth of only a single strain. The minimal inhibitory concentration

ranged from 0.9375 ± 0.44194 for Dactylorhiza hatagirea and Epigeneium amplum up

to 2.0833 ± 0.72169 mg/ml for Cymbidium iridioides and Pholidota imbricata, mak-

ing these the most and least potent extracts, respectively. Sensitivity decreased from

E. coli > B. subtilis > S. aeruginosa > P. aeruginosa > S. typhi > K. pneumonia (Sub-

edi et al. 2011).

Antibacterial activities of different extracts of epiphytic orchids (Eria spicata,

Bulbophyllum affine, Vanda cristata, Rhynchostylis retusa, Dendrobium nobile, Dendrobium amoenum, Coelogyne cristata, Pholidota imbricata) were tested against

5 species of bacteria (Gram-positive bacteria S. aureus and Bacillus subtilis and

Gram-negative bacteria Vibrio cholerae, E. coli and Klebsiella pneumonia) and anti-

fungal activities were tested against 3 species of fungi (Candida albicans, Rhyzopus

stolonifer and Mucor spp.) (Marasini and Joshi 2012). Only Coelogyne stricta (leaf)

and Dendrobium amoenum were shown good activity against Klebsiella pneumonae

but Pholidota articulata and P. imbricata have showed good activity against E. coli.

Eria spicata, Bulbophyllum affine, Vanda cristata and Rhynchostylis retusa have

showed weak activity against all bacteria. Dendrobium nobile displayed good inhibi-

tion against S. aureus but weak one against all other bacteria. All plants extract ex-

hibited very good inhibition against S. aureus. Pholidota imbricata and C. cristata

have showed the highest inhibition against V. cholera and S. aureus respectively. Af-

ter evaluating the zone of inhibition values of various extracts, two extracts with

highest zone of inhibition value were taken for MIC test by two fold serial dilution

method. MIC of extract of C. cristata was tested against S. aureus and that of P. im-

bricata against V. cholerae. The MIC of C. cristata against S. aureus was observed

at 31.25 mg/ml and that of P. imbricata against V. cholerae was observed at 62.5

mg/ml. The minimum bactericidal concentration (MBC) values of the extracts of

P. imbricata against V. cholerae and C. cristata against S. aureus were observed at

125 mg/ml and 250 mg/ml respectively. Coelogyne stricta (pseudobulb), C. stricta

(leaf) and Dendrobium amoenum exhibited no activity while P. imbricata and P. ar-ticulata extracts revealed good activity against fungal organisms. Rest of the extracts

47

showed moderate or even very weak activity against selected fungal pathogens. An-

tibacterial activities shown by C. cristata, P. articulata and P. imbricata extracts

were more active than other extracts. P. imbricata and P. articulata extracts have

found intermediate activity but all others extracts have showed very less activity or

even failed to show activity against all different five fungal organisms (Marasini and

Joshi 2012).

Rashmi and co-workers (2015) have determined antibacterial and radical scav-

enging activity of extracts of four epiphytic orchids, namely Luisia zeylanica,

Pholidota pallida, Dendrobium nutantiflorum and Coelogyne breviscapa collected at

different places of Western Ghats of Karnataka, India. Antibacterial and radical

scavenging activity of orchid extracts was determined by Agar well diffusion and

DPPH free radical scavenging activity. Two Gram-positive bacteria viz., B. subtilis

and B. coagulans and two Gram-negative bacteria viz., E. coli and Salmonella typhi

were used. In study of Rashmi and co-workers (2015), it was observed that the ex-

tracts of all orchids exhibited inhibitory effect against all tested bacteria, but to

a varied extent. Among orchids, marked antibacterial activity was displayed by

C. breviscapa as indicated by wider zones of inhibition. Other orchids exhibited

more or less similar inhibitory activity against test bacteria. Among Gram-positive

and Gram-negative bacteria, B. subtilis and E. coli were inhibited to higher extent by

extract of orchids respectively. Extracts were shown to inhibit B. coagulans and

S. typhi to more or less similar extent. Moreover, all orchid extracts displayed dose

dependent scavenging of radicals i.e., on increasing the concentration of extract,

scavenging efficacy was also increased. Highest and least scavenging potential was

exhibited by extract of L. zeylanica and P. pallida respectively as revealed by IC50

values. The scavenging effect of orchid extracts is in the order – L. zeylanica (62.62

µg/ml) > C. breviscapa (71.91 µg/ml) > D. nutantiflorum (91.46 µg/ml) > P. pallida

(128.31 µg/ml). Reference antioxidant i.e., ascorbic acid displayed higher scaveng-

ing activity (1.12 µg/ml) when compared to orchid extracts. The observed bioactivi-

ties of orchid extracts could be ascribed to the presence of phytochemicals, in par-

ticular phenolic compounds. In study of Rashmi and co-workers (2015), a positive

correlation between the phenolic content and radical scavenging activity of orchid

extracts was observed, i.e., extracts possessing high phenolic content exhibited

marked scavenging of DPPH free radicals.

Aqueous, ethanolic and chloroform soluble extracts of leaf and pseudobulb of

Bulbophyllum neilgherrense (5.50 w/v) were screened for their antibacterial poten-

tial against five species of bacteria, viz., E. coli, S. aureus, B. pumilus, P. aeruginosa

and P. putida (Priya and Krishnaveni 2005). The zone of inhibition was the largest

when alcoholic extract was used. Results of Priya and Krishnaveni (2005) revealed

that the extracts, though less effective than streptomycin, proved inhibitory to bacte-

rial growth in vitro. The growth inhibitory effect was more pronounced in ethanolic

extracts of leaf and pseudobulb followed by chloroform and aqueous extracts. The

ethanolic leaf extract was found more effective against E. coli, S. aureus and

P. aeruginosa than B. pumilus and P. putida. The pseudobulb extract was found

more effective against P. aeruginosa and P. putida (Priya and Krishnaveni 2005).

In our previous study (Buyun et al. 2016, Tkachenko et al. 2015, 2016, Góralczyk

et al. 2016), we have reinforced the assumption that Coelogyne species could be poten-

48

tial antibacterial or antifungal resource. We have determined antifungal potential of

eleven species of orchids namely Coelogyne viscosa Lindl., C. cristata Lindl., C. law-

renceana Rolfe, C. pandurata Lindl., C. assamica Linden & Rchb.f., C. fimbriata

Lindl., C. ovalis Lindl., C. asperata Lindl., C. speciosa (Blume) Lindl., C. tomentosa

Lindl., and C. brachyptera Rchb.f. against Candida albicans. Ethanolic orchid extracts

resulted in considerable suppression of growth of C. albicans. The orchid extracts from

various species of Coelogyne genus displayed varied antifungal potency. Among or-

chids selected, marked antifungal efficacy was observed in case of C. speciosa (mean

diameter of growth of inhibition zones was 19.7 mm), C. ovalis (18.2 mm), C. brachyp-tera (17.2 mm), and C. assamica (17.1 mm). Extract of C. cristata displayed least in-

hibitory activity against test fungus (mean diameter of growth of inhibition zones was

14.0 mm). Orchids were shown to exhibit antifungal activity against a variety of mold

species. Our results showed that different extracts of epiphytic orchids from Coelogyne

genus have potent antifungal properties against Candida albicans. Antifungal activities

shown by C. speciosa, C. ovalis, C. brachyptera, and C. assamica extracts were most

active then other extracts (Tkachenko et al. 2015). Moreover, ethanolic orchid leaf ex-

tracts resulted in considerable suppression of growth of S. aureus. The orchid extracts

from various species of Coelogyne genus displayed varied antimicrobial potency.

Among orchids selected, marked antimicrobial efficacy was observed for C. cristata

(mean diameter of growth of inhibition zones was 27.5 mm), C. tomentosa (26 mm),

C. lawrenceana (26 mm), C. brachyptera (26 mm), C. viscosa (25.5 mm), C. pandura-

ta (24.5 mm), and C. fimbriata (24 mm). Thus, our results showed that different ex-

tracts of epiphytic orchids from Coelogyne genus have potent antimicrobial properties

against S. aureus (Tkachenko et al. 2016).

Previously, we have investigated the antibacterial effects of ethanolic extract of

Coelogyne brachyptera leaves against specific Gram-positive (Staphylococcus aure-

us ATCC 25923 and methicillin-resistant S. aureus locally isolated) and Gram-

negative bacteria (Pseudomonas aeruginosa ATCC 27853 and Escherichia coli

ATCC 25922) and fungal strain (Candida albicans) (Buyun et al. 2016a). Our results

showed that the ethanolic extract of C. brachyptera leaves showed strong activity

against the Gram-positive bacterial strains (20 mm diameter of inhibition zone for

S. aureus and 26 mm for meticillin-resistant S. aureus), and moderate activity

against the Gram-negative bacteria (14 mm for E. coli and 11 mm for P. aerugino-

sa). Extract of C. brachyptera has displayed strong inhibitory activity against test

fungus Candida albicans (mean diameter of inhibition zone was 17.2 mm). Our

study revealed the good antimicrobial and antifungal activities of ethanolic leaf ex-

tracts of C. brachyptera. Yet, this research illustrates that a Gram-positive bacterium

was more susceptible to the ethanolic leaf extracts of C. brachyptera as compared to

Gram-negative bacteria species. Beside, extract of C. brachyptera leaves has dis-

played strong inhibitory effect against test fungus C. albicans (Buyun et al. 2016a). We also determined antibacterial and antifungal potential of ethanolic extract of

C. cristata leaves against Gram-positive (Staphylococcus aureus) and Gram-negative

(Pseudomonas aeruginosa and Escherichia coli) bacterial strains (Góralczyk et al.

2016). All microorganisms tested were susceptible to the leaf extract of C. cristata.

Extract of C. cristata displayed the least inhibitory activity against test fungus (mean

diameter of growth of inhibition zones was 14 mm). Our results showed that the etha-

49

nolic extract of C. cristata leaves exhibited strong activity against the Gram-positive

bacterial strain (27 mm of inhibition zone diameter for S. aureus), and moderate activi-

ty against the Gram-negative bacteria (13 mm for E. coli). P. aeruginosa appeared to

be less sensitive to the extract (the inhibition zone was 10 mm). In our present study

we conclude that ethanolic extract of C. cristata leaves has potent antimicrobial activi-

ty against S. aureus (Góralczyk et al. 2016).

The ethanolic extract of C. ovalis leaves showed strong activity against S. aureus

(27 mm of inhibition zone diameter), while ethanolic extract from pseudobulbs revealed

less activity (22 mm). Methanolic and ethyl acetate extracts obtained from C. ovalis leaves also showed appreciable antimicrobial activity (32 mm and 35 mm, respectively),

whereas those extracts from pseudobulbs revealed no antibacterial activity against S. au-reus (Buyun et al. 2016c). Our study has shown that ethanolic extracts from leaves and

pseudobulbs of C. tomentosa exhibited strong activity against S. aureus (inhibition zone

diameter were 29 mm and 30 mm, respectively), while methanolic extract from leaves and pseudobulbs revealed less profound activity (18 mm and 10 mm, respectively).

Moreover, it has been observed that ethyl acetate extract obtained from C. tomentosa

pseudobulbs also showed appreciable antimicrobial activity (25 mm), while those ex-

tracts from the leaves, as well as hexane and dichloromethane extracts both from the

leaves and pseudobulbs revealed no antibacterial activity against S. aureus. Hence, the

overall results of the present investigation provide evidence that the crude extracts ob-

tained from leaves and pseudobulbs of C. tomentosa could be considered as promising

natural antimicrobial products (Buyun et al. 2016b). The ethanolic extract from leaves and pseudobulbs of C. huettneriana exhibited strong activity against E. coli (inhibition

zone diameter were 28 mm and 13 mm, respectively), while others extracts from leaves and pseudobulbs revealed minimum activity. Similarly, it has been observed that etha-

nolic extract obtained from leaves and pseudobulbs also showed appreciable antimicro-

bial activity against S. aureus (19.5 mm and 21 mm, respectively), while those extracts

in ethyl acetate, hexane and dichloromethane both from the leaves and pseudobulbs re-

vealed no antibacterial activity against S. aureus (Buyun et al. 2016d).

Also, the results obtained from the present research showed antimicrobial potential

of C. speciosa extracts against S. aureus. So these plants extracts can be used as anti-

septics and antimicrobial agents in medicine and veterinary. The antibacterial activity

in Coelogyne may be due to presence of alkaloids, bibenzyl derivatives, flavonoids,

phenanthrenes and terpenoids. Clinical studies confirmed diuretic, anti-rheumatic, an-

ti-inflammatory, anti-carciogenic, antimicrobial, anticonvulsive, relaxation, neuro-

protective and antivirus activities (Pérez Gutiérrez 2010).

Kovács and co-workers (2008) have furnishes an overview of the hydroxy or/and

methoxy-substituted 9,10-dihydro/phenanthrenes, methylated, prenylated and other

monomeric derivatives, dimeric and trimeric phenanthrenes and their biological ac-

tivities. A fairly large number of phenanthrenes have been reported from higher

plants, mainly in the Orchidaceae family, in the species of Dendrobium, Bulbophyl-

lum, Eria, Maxillaria, Bletilla, Coelogyna, Cymbidium, Ephemerantha and Epiden-

drum. A few phenanthrenes have been found in the Hepaticae class and Dioscore-

aceae, Combretaceae and Betulaceae families as well. These plants have often been

used in traditional medicine, and phenanthrenes have therefore been studied for their

cytotoxicity, antimicrobial, spasmolytic, anti-inflammatory, antiplatelet aggregation,

50

antiallergic activities and phytotoxicity (Kovács et al. 2008). Finally, it should be

noted, that occurrence of phenanthrenes and dihydrophenanthrenes in orchids is

considered as accumulation of orchid phytoalexins in response to infection of orchid

plant by mycorrhizal fungi (Fisch et al. 1973).

It has been reported that many natural products including pigments, enzymes and

bioactive components are soluble in water, which explains the highest yield of ex-

tract, while some of the solvents especially acetone are selective for tannins (Rao

2004). Different solvents have various degrees of solubility for different phytocon-

stituents (Manilal and Sathishkumar 1986). Therefore, we suggest that different an-

timicrobial activity of extracts obtained from C. speciosa diluted with various sol-

vents is determined by different agents presented in extracts.

A number of phytochemicals such as alkaloids, bibenzyl derivatives, flavonoids,

phenanthrenes etc. have been reported from orchids. Presence of these phytochemi-

cals provides antimicrobial, antitumor, anti-inflammatory, antiviral activities, etc.

A number of members of orchid family are used as potent inhibitor against bacteria

and also proved to be a potent antimicrobial agents. The methanolic extract from dif-

ferent parts of orchids has shown antimicrobial activity. A broad spectrum antibacte-

rial activity (24 bacteria and protozoan) was exhibited by Pseudovanilla foliata (syn.

Galeola foliata) leaves and stem bark extracted with petrol, dichloromethane, ethyl

acetate, butanol and methanol. A very good level of activity was demonstrated by

the dichloromethane and ethyl acetate fractions (Khan and Omoloso 2004).

Moreover, endophytic fungi isolated from different orchids could be of potential

antibacterial or antifungal resource. Antimicrobial activity of ethanolic extract of

fermentation broth of 53 endophytes (30 isolates from Dendrobium devonianum and

23 endophytic fungi from D. thyrsiflorum) was explored by Xing and co-workers

(2011) using agar diffusion test. 10 endophytic fungi in D. devonianum and 11 in

D. thyrsiflorum exhibited antimicrobial activity against at least one pathogenic bac-

terium or fungus among 6 pathogenic microbes (E. coli, B. subtilis, S. aureus, Can-

dida albicans, Cryptococcus neoformans, and Aspergillus fumigatus). Out of the

fungal endophytes isolated from D. devonianum and D. thyrsiflorum, Phoma dis-

played strong inhibitory activity (inhibition zones in diameter > 20 mm) against

pathogens. Epicoccum nigrum from D. thyrsiflorum exhibited antibacterial activity

even stronger than ampicillin sodium. Fusarium isolated from the two Dendrobium

species was effective against the pathogenic bacterial as well as fungal pathogens

(Xing et al. 2011). Vaz and co-workers (2009) have examine antimicrobial activity

of endophytic fungi isolated from the leaves, stems, and roots of 54 species of Or-

chidaceae collected in a Brazilian tropical ecosystem. In total, 382 filamentous fungi

and 13 yeast isolates were obtained and cultured to examine the production of crude

extracts. Thirty-three percent of the isolates displayed antimicrobial activity against

at least one target microorganism. The extracts of endophytic fungi isolated from

leaves of terrestrial orchids in semideciduous forest were more active against E. coli,

whereas extracts of endophytic fungi from roots of rupicolous orchids collected in

rock fields were more active against Candida krusei and Candida albicans. Among

the fungi that were screened in the study, 22 isolates held their antimicrobial activi-

ties after replication and were therefore selected for assessment of the MIC, which

ranged from 62.5 to 250 µg/ml and 7.8 to 250 µg/ml against bacteria and fungi, re-

51

spectively. One isolate of Alternaria sp. and one isolate of Fusarium oxysporum pre-

sented the strongest antibacterial activity. Three Fusarium isolates, Epicoccum

nigrum, and Sclerostagonospora opuntiae showed the greatest MIC values against

the pathogenic yeasts (Vaz et al. 2009). The work also signifies the earlier findings

on various antimicrobial activity of orchid (Pérez Gutiérrez 2010) and validates the

findings that the presence of phytochemicals in orchid may be the reason for this ef-

ficacy.

CONCLUSIONS

The examined ethanolic extracts obtained from leaves and pseudobulbs of C. spe-

ciosa showed different antibacterial activities against S. aureus and E. coli, while

methanol, ethyl acetate, hexane, and dichloromethane extracts had no antibacterial ac-

tivities. The ethanolic extracts from leaves exhibited notable antimicrobial activity

against S. aureus and E. coli (inhibition zone diameter were 21.5 and 20 mm). The ob-

served activity may contribute to the reasons why the leaves are used for infectious

and inflammatory conditions in ethnomedicine. The research showed that ethanolic

extracts of C. speciosa possess antibacterial potency against S. aureus and E. coli. These findings led the authors to suggest that these extracts may be used as a natural

antiseptics and antimicrobial agents in medicine and veterinary practice. Nevertheless,

despite the promising results, more research should be carried out to further evaluate

the roles of particular compounds, isolated from all parts of orchid plants, attributable

to antimicrobial activity.

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SUMMARY

Orchids have been used all over the world in traditional healing and treatment

system of a large number of diverse diseases. Extracts and metabolites of these

54

plants demonstrate useful pharmacological activities, i.e. diuretic, antirheumatic, an-

ti-inflammatory, anticarcinogenic, hypoglycemic, antimicrobial, anticonvulsive, re-

laxation, neuroprotective, and antivirus, activities (Pérez Gutiérrez 2010). Phyto-

chemical studies in the genus Coelogyne revealed that these plants have a wide array

of secondary compounds resulting in profound biological activity, antimicrobial, in

particular (Pant 2013, Sahaya et al. 2013). In this regard, it is important to investi-

gate scientifically those plants which have been used in traditional medicines as po-

tential sources of novel antimicrobial compounds. So the present study was conduct-

ed to investigate in vitro antimicrobial activity of various extracts obtained from

vegetative parts of Coelogyne speciosa against Gram-positive (Staphylococcus aure-

us ATCC 25923) and Gram-negative bacteria (Escherichia coli ATCC 25922). The

leaves and pseudobulbs of C. speciosa plants, cultivated under glasshouse condi-

tions, were sampled at M.M. Gryshko National Botanical Garden (NBG), National

Academy of Science of Ukraine. Freshly leaves and pseudobulbs were washed,

weighted, crushed, and homogenized in 96% ethanol, methanol, ethyl acetate, hex-

ane, and dichloromethane (in proportion 1 : 19) at room temperature. Antimicrobial

activity was determined using the agar disk diffusion assay (Bauer et al. 1966). The

present study has shown that ethanolic extracts from leaves and pseudobulbs of C.

speciosa exhibited strong activity against S. aureus (inhibition zone diameter were

21.5 mm and 19 mm, respectively), while methanolic extract from leaves and pseu-

dobulbs revealed mild activity (8.1 and 8 mm). Moreover, it has been observed that

ethyl acetate, hexane and dichloromethane extracts obtained from leaves and pseu-

dobulbs of C. speciosa revealed no antibacterial activity against S. aureus. Our re-

sults also showed that ethanolic extract from leaves of C. speciosa exhibited strong

activity against E. coli (inhibition zone diameter was 21 mm), while others extracts

from pseudobulbs revealed minimum activity (inhibition zone diameter was 12

mm). Those extracts in ethyl acetate, hexane and dichloromethane both from the

leaves and pseudobulbs revealed no antibacterial activity against S. aureus. The an-

tibacterial activity in Coelogyne may be due to presence of alkaloids, bibenzyl de-

rivatives, flavonoids, phenanthrenes and terpenoids. Clinical studies confirmed diuret-

ic, anti-rheumatic, anti-inflammatory, anti-carcinogenic, antimicrobial, anticonvulsive,

relaxation, neuro-protective and antivirus activities (Pérez Gutiérrez 2010). The ob-

served activity may contribute to the reasons why the leaves are used for infectious

and inflammatory conditions in ethnomedicine. Thus, the research showed that etha-

nolic extracts of C. speciosa possess antibacterial potency against S. aureus and

E. coli and may be used as a natural antiseptics and antimicrobial agents in medicine

and veterinary practice. Considering the medicinal importance of the tested microor-

ganisms, the findings of this research are considered to be very promising in the per-

spective of new drug development from plant sources.